Autophagy is a catabolic process performed by eukaryotic cells in order to maintain homeostasis and to degrade unwanted or toxic cellular content such as misfolded proteins. The classical autophagy pathway in mammals is depicted in FIG. 1A. Observations indicate a critical role of autophagy in cancer cells e.g. survival of colorectal tumors is abolished when treated with autophagy inhibitors. However, the exact role of autophagy in cancer is very complex and highly debated. On the one hand, autophagy has been associated with increased survival of tumors, for example, under hypoxic or nutrient deprivation conditions; on the other hand, autophagy prevents DNA damage in normal cells, and therefore can serve a tumor suppressing role within the cell.
Many autophagy inhibitors such as 3-methyladenine (3-MA) or hydroxychloroquine (CQ) are promiscuous compounds. Moreover, many FDA-approved drugs interact with multiple protein targets Inhibition of mTOR through rapamycin leads to an activation of autophagy, while inhibition of VPS34 by Wortmannin prevents autophagy (FIG. 1A). In addition, the protein targets mTOR or Beclinl are effectors of multiple pathways and are therefore pleiotropic by nature.
Current intervention methods targeting the autophagy pathway are somewhat indirect and intervene very early in the pathway, which may allow the cell to regulate the autophagy pathway through other checkpoint events. Recently, a research group at Sanofi recognized the lack of direct autophagy inhibitors and its negative impact on the cancer field. They utilized an assay comprising a fusion construct expressing enhanced green fluorescent protein (EGFP)-LC3 in two tumor cell lines and monitored the granular fluorescence in the presence of small molecules. Hence, they were able to directly measure the effect of the small molecules on the lipidation state of EGFP-LC3, using loss of granulosity as an indicator of the unlipidated form. This assay was followed with a second assay which looked specifically at the induction of cell death under starvation conditions in the presence of the small molecules identified in the primary screen. Both can be used to screen novel compounds for inhibition of LC3/Atg3 interaction.
As such, there still exists an unmet need for novel compounds which can act as direct autophagy inhibitors and their use in the study and treatment of cancer.